1. Protein-RNA complex Protein-DNA complex (70 NMR structures) Protein-RNA complex (14 NMR structures)

2. 5’ RNA Pol 5’ DNA - RNA packaging, stability - 5’ capping - splicing - 3’ end processing (cleaveage and polyadenylation) - export - translation mRNA, rRNA RNA Pol 5’ 3’ RNA-binding proteins RNA editing Alternative-splicing

3. 1992 HIV tar-R 1995 BIV TAR-tart 1996 HIV RRE-rev U1A-3’UTR 199819992000 20012002 N-Box B  iv nuc-SL3 L25-5SRNA S30-5SRNA 2 U1A-3’UTR Nucleolin-RNA staufen-RNA SF1-RNA Lict-RNA NMR History of protein-RNA complex

4. Different exchange rates relative to the chemical shifts difference  k off  k off Fast exchange slow exchange Intermediate exchange

5. Different exchange rates relative to the chemical shifts difference  k off slow exchange Kd < 10 -9 M (1 nM)  k off < 10 -1 s -1 ( 0.1 Hz)  k off Fast exchange Kd > 10 -5 M (10  M)  k off > 10 3 s -1 ( 1000 Hz) Intermediate exchange 10 nM < Kd < 1  M At 600MHz,  is between 6 and 600 Hz (0.01 to 1 ppm)

6. nPTB-34 + 2 CUCUCU Kd= 10-70  M [nPTB-34(CUCUCU) 2 ]

7. 15N-TROSYs of nPTB-34 binding CUCUCU Recorded at 313K on a Bruker Avance-900 spectrometer Kd= 10-70  M

8. x x 15N-TROSYs of nPTB-34 binding CUCUCU recorded at 313K on a Bruker Avance-900 spectrometer blue: free red: bound 2:1 free 1:1

9. RRM3 RRM4 1 H, 15 N chemical shift difference between PTB34 free and PTB34 bound

10. Major chemical shift changes in PTB-34 Structure from Conte et al, EMBO (2000) RNA

11. staufen + RNA Kd= 0.1  M [staufen-RNA] Ramos et al, EMBO J, 2000

12. Intermediate exchange Ramos et al, EMBO J, 1999 Free protein1:0.5Complex 1:1

13. Slow exchange free 0:1 bound 0.5:1 1:1

14. Adding UGCAUGU to Fox1 free protein fully saturated protein Fox1-UGCAUGU is in slow exchange

15. fox1 free Fox1 + UGCAUGU 1 H- 15 N 2D HSQC (900 MHz)

16. UGCAUGU UGCAUGU Fox1 + UGCAUGU 1 H- 1 H 2D TOCSY

17. Characterizing the protein-RNA interface The protein-RNA intermolecular NOEs

18. RNA 2D NOESY (D2O) Protein aliphatic side chains Protein aliphatic side chains Y, F

19. NOEs showing intermolecular stacking C10C11 Y140 R127     Y58 R49 G13   1’ 

20. Intermolecular NOEs to the stem T52 U17 V27 G16 A7 A15 A14 A6 A8

21. 13 C proteinRNA protein 13 C RNA proteinRNA Intermolecular NOEs

22. Inter/intra molecular NOEs 1H1H 13 C decouple   x-xx mm t1 t2  = 1/2 1 J H-C=3 ms  m = NOE mixing time 50-200ms Otting and Wuthrich, Quat. Review. Biophysics (1990)

23. Inter/intra molecular NOEs IH H I IH H I IH H I IH H I 180-180 180-0 0-00-0 0-180

24. Intramolecular NOEs IH H I + ++= - -+= IH H I

25. Intermolecular NOEs IH H I + --= - +-= IH H I

26. 2D 1 H- 1 H Filtered NOESY Peterson et al, JBNMR 2004

27. 2D NOESY PTB RRM 3 & 4 (H88A) with CUCU Intermolecular NOEs 13 C protein- 12 C RNA

28. 1 H- 13 C HSQC of CUCU (sugars labeled) (provided by Prof. S.Pitsch EPFL) Free CUCU CUCU-RRM1 CUCU-RRM4 C1’-H1’ C4’-H4’ C2’-H2’ C3’-H3’ C5’-H5’,H5”

29. 2D NOESY PTB RRM 3 & 4 (H88A) with CUCU Intermolecular NOEs 13 C protein- 12 C RNA Intermolecular NOEs 12 C protein- 13 C RNA sugars

30. RBD2 RBD1 5’ 3’ CC U G A AA AUG GA 5 10 15 150 intermolecular nOes C

31. RBD2 RBD1 5’ 3’ Precision of the RDB12-RNA complex linker 19 structures bb rmsd 1.56 ± 0.40 Å

32. RBD2-RNA-RBD1 “sandwich” 5’ 3’ 1 G16 22 F56 5’ 3’ RBD2 RBD1 linker Allain et al, EMBO J (2000)